Formulations comprising antisense nucleotides to connexins

ABSTRACT

A therapeutic and/or cosmetic formulation comprising at least one anti-sense polynucleotide to a connexin protein together with a pharmaceutically acceptable carrier or vehicle is useful in site specific down regulation of connexin protein expression, particularly in reduction of neuronal cells death, wound healing, reduction of inflammation, decrease of scar formation and skin rejuvenation and thickening.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. application Ser. No.12/592,668, filed Nov. 30, 2009, which is a continuation of U.S.application Ser. No. 11/447,599 filed on Jun. 5, 2006, which is acontinuation of U.S. application Ser. No. 09/890,363, filed Jul. 27,2001 (now issued as U.S. Pat. No. 7,098,190), which is a U.S. NationalStage Application of International PCT Application No. PCT/GB00/00238,filed Jan. 27, 2000 (published as WO00/44409 on Aug. 3, 2000) and claimsthe benefit of priority to NZ 333928 (filed Jan. 27, 1999) and NZ 500190(filed Oct. 7, 1999). The contents of each of which are herebyincorporated in their entireties.

FIELD

This invention relates to formulations for use in therapeutic and/orcosmetic treatments, particularly those in which a localised disruptionin direct cell-cell communication is desirable.

BACKGROUND

Gap junctions are cell membrane structures which facilitate directcell-cell communication. A gap junction channel is formed of twohemichannels (connexons), each composed of six connexin subunits. Theseconnexins are a family of proteins, commonly named according to theirmolecular weight or classified on a phylogenetic basis ie. into an αclass and a β class.

An ability to control connexin expression (and in particular todownregulate it) would therefore provide an opportunity to modulatecell-cell communication within a patient for therapeutic and/or remedialpurposes. However, as a number of connexin proteins are expressed widelythroughout the body, a general downregulatory effect is undesirable ininducing a therapeutic effect at a specific site.

Anti-sense oligodeoxynucleotides (ODN's) have considerable potential asagents for the manipulation of specific gene expression (reviewed: Steinet al., 1992; Wagner 1994). However, there remain difficulties whichneed to be overcome. These include the short half life of such ODN's(unmodified phosphodiester oligomers typically have an intracellularhalf life of only 20 minutes owing to intracellular nuclease degradation(Wagner 1994)) and their delivery consistently and reliably to targettissues.

It was with the intent of at least partially overcoming thesedifficulties that the applicants devised the present invention.

SUMMARY OF THE INVENTION

Accordingly, in a first aspect, the invention provides a formulation foruse in therapeutic and/or cosmetic treatment, which formulationcomprises: at least one anti-sense polynucleotide to a connexin protein;together with a pharmaceutically acceptable carrier or vehicle.

In one preferred form, the formulation contains polynucleotides to oneconnexin protein only. Most preferably, this connexin protein isconnexin 43.

Many aspects of the invention are described with reference tooligodeoxynucleotides. However it is understood that other suitablepolynucleotides (such as RNA polynucleotides) may be used in theseaspects.

Alternatively, the formulation contains oligodeoxynucleotides to morethan one connexin protein. Preferably, one of the connexin proteins towhich oligodeoxynucleotides are directed is connexin 43. Other connexinproteins to which oligodeoxynucleotides are directed include connexin26, connexin 31.1 and connexin 32.

Conveniently, the oligodeoxynucleotide to connexin 43 is selected from:

(SEQ ID NO: 1) GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC;  (SEQ ID NO: 2)GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC;  and  (SEQ ID NO: 3)GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT Most conveniently, the oligodeoxynucleotide to connexin 43 is:

(SEQ ID NO: 1) GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC. Conveniently, the oligodeoxynucleotide to connexin 26 is:

(SEQ ID NO: 4) TCC TGA GCA ATA CCT AAC GAA CAA ATA. Conveniently, the oligodeoxynucleotide to connexin 31.1 is:

(SEQ ID NO: 5) CGT CCG AGC CCA GAA AGA TGA GGT C. Conveniently, the oligodeoxynucleotide to connexin 32 is:

(SEQ ID NO: 6) TTT CTT TTC TAT GTG CTG TTG GTG A. 

The anti-sense polynucleotides may be formulated for parenteral,intramuscular, intracerebral, intravenous, subcutaneous or transdermaladministration. The antisense polynucleotides are preferablyadministered topically (at the site to be treated). Suitably theantisense polynucleotides are combined with a pharmaceuticallyacceptable carrier, vehicle or diluent to provide a pharmaceuticalcomposition.

Suitable pharmaceutically acceptable carriers or vehicles include any ofthose commonly used for topical administration. The topical formulationmay be in the form of a cream, ointment, gel, emulsion, lotion or paint.The formulation of the invention may also be presented in the form of animpregnated dressing.

Suitable carrier materials include any carrier or vehicle commonly usedas a base for creams, lotions, gels, emulsions, lotions or paints fortopical administration. Examples include emulsifying agents, inertcarriers including hydrocarbon bases, emulsifying bases, non-toxicsolvents or water-soluble bases. Particularly suitable examples includelanolin, hard paraffin, liquid paraffin, soft yellow paraffin or softwhite paraffin, white beeswax, yellow beeswax, cetostearyl alcohol,cetyl alcohol, dimethicones, emulsifying waxes, isopropyl myristate,microcrystalline wax, oleyl alcohol and stearyl alcohol.

Preferably, the pharmaceutically acceptable carrier or vehicle is a gel,suitably a nonionic polyoxyethylene-polyoxypropylene copolymer gel, forexample, a Pluronic gel, preferably Pluronic F-127 (BASF Corp.). Thisgel is particularly preferred as it is a liquid at low temperatures butrapidly sets at physiological temperatures, which confines the releaseof the ODN component to the site of application or immediately adjacentthat site.

An auxiliary agent such as casein, gelatin, albumin, glue, sodiumalginate, carboxymethylcellulose, methylcellulose, hydroxyethylcelluloseor polyvinyl alcohol may also be included in the formulation of theinvention.

The pharmaceutical composition may be formulated to provide sustainedrelease of the antisense polynucleotide.

Conveniently, the formulation further includes a surfactant to assistwith oligodeoxynucleotide cell penetration or the formulation maycontain any suitable loading agent. Any suitable non-toxic surfactantmay be included, such as DMSO. Alternatively a transdermal penetrationagent such as urea may be included.

In a further aspect, the invention provides a method of site-specificdownregulation of connexin protein expression for a therapeutic and/orcosmetic purpose which comprises administering a formulation as definedabove to a site on or within a patient at which said downregulation isrequired.

In still a further aspect, the invention provides a method of reducingneuronal cell death which would otherwise result from a neuronal insultto a specific site in the brain, spinal cord or optic nerve of a patientwhich comprises the step of administering a formulation as defined aboveto said site to downregulate expression of connexin protein(s) at andimmediately adjacent said site.

Preferably, the formulation is administered to reduce neuronal loss dueto physical trauma to the brain, spinal cord or optic nerve.

Conveniently, the formulation is administered in a sufficient amount todownregulate expression of said connexin protein(s) for at least 24hours post-administration.

In yet a further aspect, the invention provides a method of promotingwound healing in a patient which comprises the step of administering aformulation as defined above to said wound to downregulate expression ofconnexin protein(s) at and immediately adjacent the site of said wound.

Usually, the wound will be the result of trauma, including burns. It mayhowever be the result of surgery.

In yet a further aspect, the invention provides a method of reducinginflammation as part of treating a wound and/or tissue subjected tophysical trauma which comprises the step of administering a formulationas defined above to or proximate to said wound or tissue.

Preferably, said wound is a burn.

Alternatively, said wound is the result of physical trauma to tissue,including neuronal tissue such as the brain, spinal cord or optic nerve.

In yet a further aspect, the invention provides a method of decreasingscar formation in a patient who has suffered a wound which comprises thestep of administering a formulation as defined above to said wound todownregulate expression of connexin protein(s) at and immediatelyadjacent the site of said wound.

Again, the wound may be the result of trauma or surgery, with theformulation being applied to the wound immediately prior to surgicalrepair and/or closure thereof.

In yet a further aspect, the invention provides a method of skinrejuvenation or thickening for a cosmetic or therapeutic purpose whichcomprises the step of administering, once or repeatedly, a formulationas defined above to the skin surface.

Conveniently, said formulation includes oligodeoxynucleotides directedto connexin 26 or connexin 43 and is administered to regulate epithelialbasal cell division and growth.

In another embodiment, said formulation includes oligodeoxynucleotidesdirected to connexin 31.1 and is administered to regulate outer layerkeratinisation.

Preferably, the formulation is a cream or gel.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 5 show sections of rat brain lesions treated with Pluronicgel containing antisense oligodeoxynucleotides specific to connexin 43,or for control lesions, Pluronic gel alone. In all cases lesions weresectioned serially in a coronal plane and the mid point sections usedfor analysis. Each image (except FIG. 5) shows 4 mm by 5.33 mm oftissue. FIG. 5 is approximately 1.2 mm by 2 mm.

FIGS. 1A and 1C show two side of a control lesion 24 hours afterlesioning. The lesion has been treated with Pluronic gel alone. Thesections have been Nissl stained (blue nuclei) and antibody labelledwith the neuronal marker Neuronal-N (brown cells). FIGS. 1B and 1D showgrey scale images of 1A and 1C respectively with the outline of thelesion marked. Note the large size of the lesion and the irregularspreading edges. The lesion has spread downwards toward the corpuscallosum (dashed line) within just 24 hours of lesioning.

FIGS. 2A-2B: A control lesion 24 hours after wounding. FIG. 2A showsNissl staining (blue nuclei) and Neuronal-N labelling of viable neurons.FIG. 2B is a grey scale equivalent with the lesion edge marked and thetop of the corpus callosum marked (dashed line). The original needletract is clear but neuronal death has occurred well back from the lesionedge as indicated by the Neuronal-N labelling. The edges of the lesionare irregular and the lesion, within just 24 hours, has spread rightdown into the corpus callosum.

FIGS. 3A and 3B are colour and grey scale images of a connexin 43antisense treated lesion, 48 hours after lesioning. The lesion outlinehas been marked on FIG. 3B to show the extent of the lesion and the topof the corpus callosum marked (dashed line). FIG. 3A has been stainedfor Nissl (blue nuclei) and Neuronal-N (pink cells). Note how compactthe lesion is, even after 48 hours, compared with control lesions (FIGS.1A-1D and 2A-2B). While there is some spread to the right hand side, theleft side of the lesion essentially follows the original needle tractwith little sign of spreading. The left side of the lesion is verystraight and it has not spread down to the corpus callosum.

FIGS. 4A and 4B show another connexin 43 antisense treated lesion 48hours after wounding. The labelling is the same as in FIG. 3A-3B withthe lesion outlined on the grey scale image (FIG. 4B). Even after 48hours this lesion is extremely compact with slight spreading only to theleft (medial side). Note how straight the right hand side of the lesionis with viable neurons right up to the edge of the needle tract (andindeed surviving within the lesioned area). The lesion is well above thecorpus callosum (dashed line) indicating virtually no downward spread.

FIG. 5: A higher magnification view showing the edge of a connexin 43antisense treated lesion. The edge of the lesion has been marked showingviable neurons (Neuronal-N labelled) right up to the edge of thewounding needle tract even 48 hours after lesioning.

FIG. 6: GFAP (red) and connexin 43 (green) immunohistochemical labellingof a connexin 43 specific antisense treated lesion, 24 hours afterlesioning. The image is taken at the lateral edge of the lesion at apoint half way down the depth of the lesion. Activated astrocyte levelsare elevated compared with controls (FIG. 7) and connexin 43 levels aremarkedly reduced. The connexin labelling remaining is generallyassociated with blood vessels (arrows).

FIG. 7: GFAP (red) and connexin 43 (green) immunohistochemical labellingof a control lesion, 24 hours after lesioning. The image is from themedial edge of the lesion and shows GFAP levels slightly elevated overunlesioned cortex. Note the extensive connexin 43 labelling, oftenco-localised with the GFAP astrocytic marker (arrows).

FIG. 8 shows a comparison of lesion cross sectional lower half areas 24hours (circles) and 48 hours (diamonds) after lesioning. The analysiswas carried out on a mid section of serially sectioned lesion cut on thecoronal plane. Lesions were assessed using Neuronal-N antibody labellingto delineate viable neurons. DB 1 treated lesions (green markers) havebeen treated with antisense oligodeoxynucleotides specific to connexin43. The gel only lesion group (red markers) also includes empty lesionswhile the HB3 group (purple markers) are treated with gel containingrandom sequence control oligodeoxynucleotides. Note that while connexin43 antisense treated lesions can be large (presumably where theantisense has not been well delivered), the smallest lesions are allconnexin 43 antisense treated. Lesions were made to a depth of 2 mm andanalysis covers 1 mm and below so as to exclude the outer edge where theantisense did not sit.

FIG. 9: Lesions in rat spinal cord 24 hours after treatment withconnexin 43 sense and antisense ODN's. The sense lesions were nodifferent from untreated controls whereas the antisense treated lesionswere smaller and with reduced inflammation.

FIG. 10: Lesions in neonatal mouse fore paws 24 hours after treatmentwith connexin 43 sense ODNs (left paw) or antisense ODNs (right paw).Note the reduction in inflammation and increased rate of healing on theantisense treated paw.

FIG. 11: Sections through the centre of the 24 hour wounds shown in FIG.10. The sections have been stained with toluidine blue to revealneutrophils. There are significantly less neutrophils in the antisensetreated wound which was also less inflamed.

FIG. 12: Pairs of rat paw lesions five days after lesioning that havebeen treated with connexin 43 specific antisense ODNs or sense controlODNs. Antisense treated lesions are healing quicker and show less signsof scarring.

FIG. 13: Pairs of rat paw lesions made at the neonate stage, and viewedhere 8 days after lesioning. Lesions were treated with connexin 43specific antisense or control sense ODN. Hair has grown and it is clearthat antisense treatment has resulted in smaller scars and less hairloss. The site of the lesion remains prominent in the sense treatedcontrol but is difficult to detect in the antisense treated limb.

DESCRIPTION OF THE INVENTION

As broadly defined above, the focus of the invention is on site-specificdownregulation of connexin expression. This will have the effect ofreducing direct cell-cell communication at the site at which connexinexpression is downregulated, which gives rise to numeroustherapeutic/cosmetic applications as described below.

The downregulation of connexin expression is based generally upon theanti-sense approach using antisense polynucleotides (such as DNA or RNApolynucleotides), and more particularly upon the use of antisenseoligodeoxynucleotides (ODN). These polynucleotides (eg. ODN) target theconnexin protein (s) to be downregulated. Typically the polynucleotidesare single stranded, but may be double stranded.

The antisense polynucleotide may inhibit transcription and/ortranslation of the connexin. Preferably the polynucleotide is a specificinhibitor of transcription and/or translation from the connexin gene,and does not inhibit transcription and/or translation from other genes.The product may bind to the connexin gene or mRNA either (i) 5′ to thecoding sequence, and/or (ii) to the coding sequence, and/or (iii) 3′ tothe coding sequence.

Generally the antisense polynucleotide will cause the expression ofconnexin mRNA and/or protein in a cell to be reduced.

The antisense polynucleotide is generally antisense to the connexinmRNA. Such a polynucleotide may be capable of hybridising to theconnexin mRNA and may thus inhibit the expression of connexin byinterfering with one or more aspects of connexin mRNA metabolismincluding transcription, mRNA processing, mRNA transport from thenucleus, translation or m RNA degradation. The antisense polynucleotidetypically hybridises to the connexin mRNA to form a duplex which cancause direct inhibition of translation and/or destabilisation of themRNA. Such a duplex may be susceptible to degradation by nucleases.

The antisense polynucleotide may hybridize to all or part of theconnexin mRNA. Typically the antisense polynucleotide hybridizes to theribosome binding region or the coding region of the connexin mRNA. Thepolynucleotide may be complementary to all of or a region of theconnexin mRNA. For example, the polynucleotide may be the exactcomplement of all or a part of connexin mRNA. However, absolutecomplementarity is not required and polynucleotides which havesufficient complementarity to form a duplex having a melting temperatureof greater than 20° C., 30° C. or 40° C. under physiological conditionsare particularly suitable for use in the present invention.

Thus the polynucleotide is typically a homologue of the mRNA. Thepolynucleotide may be a polynucleotide which hybridises to the connexinmRNA under conditions of medium to high stringency such as 0.03M sodiumchloride and 0.03M sodium citrate at from about 50 to about 60 degreescentigrade.

The polynucleotide will typically be from 6 to 40 nucleotides in length.Preferably it will be from 12 to 20 nucleotides in length. Thepolynucleotides may be at least 40, for example at least 60 or at least80, nucleotides in length and up to 100, 200, 300, 400, 500, 1000, 2000or 3000 or more nucleotides in length.

The connexin protein or proteins targeted by the ODN will be dependentupon the site at which downregulation is to be effected. This reflectsthe non-uniform make-up of gap junction(s) at different sites throughoutthe body in terms of connexin sub-unit composition. The connexin may beany connexin that naturally occurs in a human or animal. The connexingene (including coding sequence) generally has homologue with any of thespecific connexins mentioned herein, such as homology with the connexin43 coding sequence shown in Table 3 The connexin is typically an α or βconnexin. Preferably the connexin is expressed in the skin or nervoustissue (including brain cells).

Some connexin proteins are however more ubiquitous than others in termsof distribution in tissue. One of the most widespread is connexin 43.ODN's targeted to connexin 43 are therefore particularly suitable foruse in the present invention.

It is also contemplated that ODN's targeted at separate connexinproteins be used in combination (for example 1, 2, 3, 4 or moredifferent connexins may be targeted). For example, ODN's targeted toconnexin 43, and one or more other members of the connexin family (suchas connexin 26, 31.1, 32, 36, 40 and 45) can be used in combination.

Individual antisense polynucleotides may be specific to a particularconnexin, or may target 1, 2, 3 or more different connexins. Specificpolynucleotides will generally target sequences in the connexin gene ormRNA which are not conserved between connexins, whereas non-specificpolynucleotides will target conserved sequences.

The ODN's for use in the invention will generally be unmodifiedphosphodiester oligomers. They will vary in length but with a 30 mer ODNbeing particularly suitable.

The antisense polynucleotides may be chemically modified. This mayenhance their resistance to nucleases and may enhance their ability toenter cells. For example, phosphorothioate oligonucleotides may be used.Other deoxynucleotide analogs include methylphosphonates,phosphoramidates, phosphorodithioates, N3′P5′-phosphoramidates andoligoribonucleotide phosphorothioates and their 2′-O-alkyl analogs and2′-O-methylribonucleotide methylphosphonates.

Alternatively mixed backbone oligonucleotides (MBOs) may be used. MBOscontain segments of phosphothioate oligodeoxynucleotides andappropriately placed segments of modified oligodeoxy- oroligoribonucleotides. MBOs have segments of phosphorothioate linkagesand other segments of other modified oligonucleotides, such asmethylphosphonate, which is non-ionic, and very resistant to nucleasesor 2′-O-alkyloligoribonucleotides.

The precise sequence of the antisense polynucleotide used in theinvention will depend upon the target connexin protein. For connexin 43,the applicant's have found ODN's having the following sequences to beparticularly suitable:

(SEQ ID NO: 1) GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC;  (SEQ ID NO: 2)GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC;  and  (SEQ ID NO: 3)GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT 

ODN's directed to other connexin proteins can be selected in terms oftheir nucleotide sequence by any convenient, and conventional, approach.For example, the computer programmes MacVector and OligoTech (fromOligos etc. Eugene, Oreg., USA) can be used. For example, ODN's forconnexins 26, 31, 1 and 32 have the following sequences:

(SEQ ID NO: 4) 5′ TCC TGA GCA ATA CCT AAC GAA CAA ATA   (connexin 26)(SEQ ID NO: 5) 5′ CGT CCG AGC CCA GAA AGA TGA GGT C  (connexin31.1) (SEQ ID NO: 6) 5′ TTT CTT TTC TAT GTG CTG TTG GTG A   (connexin 32)

Once selected, the ODN's can be synthesised using a DNA synthesiser.

For use in the invention, the ODN(s) require site-specific delivery.They also require delivery over an extended period of time. Whileclearly the delivery period will be dependent upon both the site atwhich the downregulation is to be induced and the therapeutic effectwhich is desired, continuous delivery for 24 hours or longer will oftenbe required.

In accordance with the present invention, this is achieved by inclusionof the ODN(s) in a formulation together with a pharmaceuticallyacceptable carrier or vehicle, particularly in the form of a formulationfor topical administration.

Once prepared, the formulations of the invention have utility in anytherapeutic/cosmetic approach where a transient and site-specificinterruption in cell-cell communication is desirable. These include intreating neuronal damage in the brain, spinal cord or optic nerve (wherethe damage is to be localised as much as possible), in the promotion ofwound healing and in reducing scar formation following, for example,cosmetic surgery or burns.

In particular, topical formulations such as creams can be employed toregulate epithelial basal cell division and growth (using ODN's targetedto connexin 43) and outer layer keratinisation (using ODN's targeted toconnexin 31.1).

The antisense polynucleotides (including the ODN) may be present in asubstantially isolated form. It will be understood that the product maybe mixed with carriers or diluents which will not interfere with theintended purpose of the product and still be regarded as substantiallyisolated. A product of the invention may also be in a substantiallypurified form, in which case it will generally comprise 90%, e. g. atleast 95%, 98% or 99% of the polynucleotide or dry mass of thepreparation.

Administration

The antisense polynucleotides (including ODN's) of the invention(typically in the form of the formulation discussed herein) may thus beadministered to a human or animal in need of treatment, such as a humanor animal with any of the diseases or conditions mentioned herein. Thecondition of the human or animal can thus be improved. Thepolynucleotide and formulation may thus be used in the treatment of thehuman or animal body by therapy. They may be used in the manufacture ofa medicament to treat any of the conditions mentioned herein.

The antisense polynucleotides may be administered by typically (at thesite to be treated). Preferably the antisense polynucleotides arecombined with a pharmaceutically acceptable carrier or diluent toproduce a pharmaceutical composition. Suitable carriers and diluentsinclude isotonic saline solutions, for example phosphate-bufferedsaline. The composition may be formulated for parenteral, intramuscular,intracerebral, intravenous, subcutaneous, or transdermal administration.

The dose at which an antisense polynucleotide is administered to apatient will depend upon a variety of factors such as the age, weightand general condition of the patient, the condition that is beingtreated, and the particular antisense polynucleotide that is beingadministered. A suitable dose may however be from 0.1 to 100 mg/kg bodyweight such as 1 to 40 mg/kg body weight.

Uptake of nucleic acids by mammalian cells is enhanced by several knowntransfection techniques for example those including the use oftransfection agents. The formulation which is administered may containsuch agents. Examples of these agents include cationic agents (forexample calcium phosphate and DEAE-dextran) and lipofectants (forexample Lipofectam™ and Transfectam™).

The routes of administration and dosages described above are intendedonly as a guide since a skilled physician will be able to determinereadily the optimum route of administration and dosage for anyparticular patient and condition.

Homologues

Homology and homologues are discussed herein (eg. the polynucleotidesmay be a homologue of sequence in connexin mRNA). Such polynucleotidestypically have at least 70% homology, preferably at least 80, 90%, 95%,97% or 99% homology with the relevant sequence, for example over aregion of at least 15, 20, 40, 100 more contiguous nucleotides (of thehomologous sequence).

Homology may be calculated based on any method in the art. For examplethe UWGCG Package provides the BESTFIT program which can be used tocalculate homology (for example used on its default settings) (Devereuxet al (1984) Nucleic Acids Research 12, p 387-395). The PILEUP and BLASTalgorithms can be used to calculate homology or line up sequences(typically on their default settings), for example as described inAltschul S. F. (1993) J Mol Evol 36: 290-300; Altschul, S, F et al(1990) J Mol Biol 215: 403-10.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/) This algorithm involves first identifyinghigh scoring sequence pair (HSPs) by identifying short words of length Win the query sequence that either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighbourhood word scorethreshold (Altschul et al, supra). These initial neighbourhood word hitsact as seeds for initiating searches to find HSPs containing them. Theword hits are extended in both directions along each sequence for as faras the cumulative alignment score can be increased. Extensions for theword hits in each direction are halted when: the cumulative alignmentscore falls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T and X determinethe sensitivity and speed of the alignment. The BLAST program uses asdefaults a word length (W) of 11, the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919)alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e. g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a sequenceis considered similar to another sequence if the smallest sumprobability in comparison of the first sequence to a second sequence isless than about 1, preferably less than about 0.1, more preferably lessthan about 0.01, and most preferably less than 0.001.

The homologous sequence typically differs from the relevant sequence byat least (or by no more than) 2, 5, 10, 15, 20 more mutations (which maybe substitutions, deletions or insertions). These mutations may bemeasured across any of the regions mentioned above in relation tocalculating homology.

The homologous sequence typically hybridises selectively to the originalsequence at a level significantly above background. Selectivehybridisation is typically achieved using conditions of medium to highstringency (for example 0.03M sodium chloride and 0.03M sodium citrateat from about 50° C. to about 60° C.). However, such hybridisation maybe carried out under any suitable conditions known in the art (seeSambrook et al (1989), Molecular Cloning: A Laboratory Manual). Forexample, if high stringency is required, suitable conditions include0.2×SSC at 60° C. If lower stringency is required, suitable conditionsinclude 2×SSC at 60° C.

Various aspects of the invention will now be described with reference tothe following experimental section which will be understood to beprovided by way of illustration only and not to constitute a limitationon the scope of the invention.

Immunomodulatory Oligonucleotides

In another embodiment, the anti-connexin antisense oligonucleotides ofthis disclosure may also have immunomodulatory activity, for example,immunostimulatory activity. In some embodiments, the oligonucleotidesmay contain 2 or more CpG sequence motifs. In some embodiments, theoligonucleotides may contain 2, 3, 4, 5, 6, 7, 8, 9, 10 or more CpGsequence motifs. In some embodiments, the oligonucleotides will containat least 1 CpG sequence motif. The oligonucleotides may have at least70%, 80%, 87%, 90% or 92% homology with SEQ ID NO:1 or SEQ ID NO:2.Exemplary anti-connexin 43 oligonucleotide sequences comprising CpGsequence motifs and having homology to SEQ ID NO: 1 and SEQ ID NO:2 areset forth below:

(SEQ ID NO. 13) CGTAATTGCGGCAGGAGGAATTGTTTCTGTCG (SEQ ID NO. 14)CGTAATTGCGGCAAGAGGAATTGTTTCTGTCG

In addition, the anti-connexin antisense oligonucleotides of thisdisclosure may contain two or more substitutions in SEQ ID NO:13 or SEQID NO:14, as shown above each of the positions at which a substitutioncan be made:

For example, in the upper sequence (SEQ ID NO: 13), the A at position 13and the T at position 28 may be substituted with either C or G. Inaddition, The T at position 7, the G at position 10, the G at position14, the A at position 16, the G at position 17, and/or the T at position22 may be each be substituted with a C. In some embodiments, theimmunomodulatory oligonucleotide may contain 3, 4, 5, 6, 7, 8 or 9 CpGsequence motifs within the sequence.

As another example, in the lower sequence (SEQ ID NO. 14), the A atposition 13 can be substituted with G, and the T at position 28 may besubstituted with either C or G. In addition, The T at position 7, the Gat position 10, the G at position 14, the A at position 16, the G atposition 17, and/or the T at position 22 may be each be substituted witha C. In some embodiments, the immunomodulatory may contain 3, 4, 5, 6,7, or 8 CpG sequence motifs within the sequence.

The anti-connexin antisense oligonucleotides of this disclosure may alsocontain two or more of the following substitutions shown above thesequences in SEQ ID NO: 15 and SEQ ID NO: 16 (substitutions are shownabove each position at which a substitution can be made):

For example, in the upper sequence (SEQ ID NO:15), the A at position 12can be substituted with G, and the T at position 27 may be substitutedwith either C or G. In addition, The T at position 6, the G at position9, the G at position 13, the A at position 15, the G at position 16,and/or the T at position 21 may be each be substituted with a C. In someembodiments, that oligonucleotide may contain 1, 2, 3, 4, 5, 6, or 7 CpGsequence motifs within the sequence. In some embodiments, theimmunomodulatory oligonucleotide may contain 2 or more CpG sequencemotifs.

As another example, in the lower sequence (SEQ ID NO:16), the A atposition 12 and the T at position 27 may be substituted with either C orG. In addition, The T at position 6, the G at position 9, the G atposition 13, the A at position 15, the G at position 16, and/or the T atposition 21 may be each be substituted with a C. In some embodiments,that oligonucleotide may contain 1, 2, 3, 4, 5, or 6 CpG sequence motifswithin the sequence. In some embodiments, the immunomodulatoryoligonucleotide may contain 2 or more CpG sequence motifs.

The immunomodulatory oligonucleotides of this disclosure may beformulated into pharmaceutical compositions according to any of thecompositions using any of the methods and pharmaceutical carriers setforth herein. The immunomodulatory oligonucleotides may also beadministered according to any method described herein. In someembodiments, the immunomodulatory oligonucleotides may be administeredto a patient in need thereof. The immunomodulatory oligonucleotides mayalso comprise modified backbones, as described herein.

The immunomodulatory oligonucleotide may be used for example, in thetreatment of wounds. In some embodiments, the immunomodulatoryoligonucleotide may be used in cosmetic treatments.

EXPERIMENTAL Experiment 1

Materials and Methods

Antisense Application

30% Pluronic F-127 gel (BASF Corp) in phosphate buffered saline(molecular grade water) was used to deliver unmodified a1 connexin(connexin 43) specific anti-sense ODN's to the developing chick embryo(Simons, et al., 1992). Chick embryos were incubated at 38° C. andstaged according to Hamilton and Hamburger stages. Eggs were windowedand the vitleline and amniotic membranes over the area to be treatedwere opened using fine forceps. After anti-sense application eggs weresealed with tape and replaced in the incubator for 48 hours at whichtime most experiments were analysed, the exception being for the timecourse analysis of a1 connexin “knockdown” and recovery.

Pluronic gel is liquid at low temperatures, 0-4 C, but sets when droppedonto the embryo at physiological temperature, remaining in place for atleast 12 hours. The gel has the additional advantage of being a mildsurfactant and this, used either alone or in conjunction with DMSO,appeared to markedly expedite ODN penetration into cells (Wagner, 1994).Addition of an FITC tag to DB1 ODN, viewed using confocal laser scanningmicroscopy, demonstrated intracellular penetration of the probes.Sequences of deoxyoligonucleotides used are shown in Table 1.

Table 1: The Effect on Limb Development of ODN Application BetweenStages 8 & 14 of Chick Embryo Development

Antisense Oligodeoxynucleotides to Connexin 43

DB1  (SEQ ID NO: 2) GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC  CG 1 (SEQ ID NO: 3) GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT 

Control Oligodeoxynucleotides

DB 1 (sense)  (SEQ ID NO: 7) GAC AGA AAC AAT TCC TCC TGC CGC AAT TAC DB 1 (chick)  (SEQ ID NO: 8) GTA GTT ACG ACA GGA GGA ATT GTT CTC GTC CV3 (random)  (SEQ ID NO: 9) TCG AAC TGT CAA GAC TGC TAT GGC GAT CAT 

Gel Only

All ODN's were applied at 0.5-1.0 μM final concentration following dosedependent analysis during preliminary experiments covering a range ofconcentrations from 0.05 μM to 50 μM. General toxicity effects onlybecame apparent with ODN concentrations greater than 10 μM. ODN gelmixtures were prepared from concentrated stock solutions stored at −80°C.

Anti-Sense Sequences

DB1 is a mouse anti-sense sequence, complementary to bases 1094-1123 ofthe a1 connexin gene. It has four mismatches with chick a1 connexinsequence. CG1 is complementary to chick a1 connexin bases 720-749.Efficacy of this probe was improved with 1% Dimethylsulphoxide (DMSO)added to the gel. DMSO had no added effect on other anti-sense ODN orcontrol results.

Control Sequences

DB1 (Chick) is the chick a1 connexin equivalent of DB1 matching chick a1connexin bases 954-983. Analysis however, indicates a high probabilityof forming stem loop structures (G=−7.0 kcal/mol, Loop Tm=92°) andhomodimerisation (Tm=1.5°) and therefore acts as a control sequence. Ithas been reported that some sense oligonucleotides can form stable DNAtriplets (Neckers et al., 1993) inhibiting transcription. However, thiswas not apparent with DB1 (sense). A random control sequence with nostable secondary structure (G=1.4 kcal/mol) and unstablehomodimerisation was also used, called CV3. An additional controlapplying equal concentration mixture of DB1 and DB1 (sense) gavebackground levels of defects.

Monitoring of Protein Knockdown

Immunohistochemical localisation of a1 connexin gap junction protein atcell-cell interfaces provides a direct measurement of the anti-senseeffect. Anti-peptide a1 connexin specific antibody probes were used tostain wholemount embryos and the connexin distribution was analysedusing confocal laser scanning microscopy according to establishedprocedures (Green et al., 1995). Control labelling for two otherconnexins expressed in the developing chick embryo (connexins b1 & b2)was similarly carried out, also using sequence specific antibodies(Becker et al., 1995).

Results

Reduction of a1 Connexin Expression

Using Pluronic F-127 gel to deliver unmodified a1 connexin specificantisense ODN's to the developing chick embryo, protein expression canbe interfered with at chosen time points and allows the anti-sensetreatment to be targeted to specific regions of a chick embryo. Adroplet of gel containing the anti-sense at a relatively lowconcentration was placed precisely onto individual embryos. The gel setsand remains in place for at least 12 hours and thus a sustained low doseof antisense is maintained in this region. The anti-sense applicationswere targeted and timed to block junction formation prior to the periodsof elevated expression in the limb, neural tube and face. These timeswere chosen to optimise the effects of the anti-sense by reducing theexpression of new protein rather than being dependent upon the turnoverof protein already in the membranes of the cells of the target tissue.Both DB1 and CG1 ODN's reduced expression of a1 connexin protein withintwo hours in the neural tube and limb bud, dramatic within 4-8 hours andpersisted at 18-24 hours and 48 hours in some tissues (data not shown).No down regulation of a1 connexin protein was evident in any of thecontrols used. Equally, two other members of the connexin familyexpressed in the chick embryo, b1 connexin and b2 connexin, wereunaffected by the a1 connexin specific anti-sense ODN.

Several parallel controls were run with all of the experiments. Theseincluded; DB1 sense, DB1 anti-sense and DB1 sense combined, DB1 chick(which forms stem loop structures with itself), random ODN's CV3,Pluronic gel alone, Pluronic gel with DMSO and PBS alone). None of thecontrols had a noticeable effect on a1 connexin protein expression.

Experiment 2

Introduction

Astrocytes constitute the most abundant cell type in the mammalianbrain. They are extensively coupled to one another and to neuronsthrough gap junctions composed predominantly of connexin 43 (Giaume andMcCarthy (1996)). Following ischaemia induced or physical brain damagethese channels remain open and a spreading wave of depression (initiatedby raised interstitial potassium and glutamate and apoptotic signals) ispropagated (Cotrina et al., (1998); Lin et al (1998)). Waves ofincreased cytosolic calcium and second messenger molecules such as IP3are slowly spread via the gap junction channels to neurons beyond thecore of the damaged region, resulting in lesion spread in the 24-48hours following the insult. In this manner, undamaged neighbouring cellsare destroyed (Lin et al., 1998), the so-called bystander effect.

This experiment investigates the ability of the formulations of theinvention to prevent this bystander effect.

Materials

Oligodeoxynucleotides were prepared with the following sequences:

(SEQ ID NO: 2) GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC  (connexin 43) (SEQ ID NO: 10) TTG TGA TTT ATT TAG TTC GTC TGA TTT C  (random control)

Methods

Oligodeoxynucleotides (ODN's)

Oligodeoxynucleotides (ODN's)

Unmodified ODN's were delivered in Pluronic F-127 gel (BASF, Germany) inphosphate buffered saline (PBS). Pluronic gel is liquid at lowtemperatures (0-4° C.) and sets at physiological temperatures, and isalso a mild surfactant. Unmodified ODN's normally have a half life ofapproximately 20 min in cells (Wagner, 1994) but the Pluronic gelloading method provides a continual diffusion source, the gel acting asa reservoir Becker et al., (1999)). ODN's specific to connexin 43 wereapplied, or control random ODN's of similar base composition, at 2 μMfinal concentration. Gel only controls were also carried out. ODN's were30 mers analysed to show that no hairpin looping or homodimerisationshould occur.

Lesioning

Brain lesions were carried out on 250-300 g male Wistar rats. Animalswere anaesthetised with 1-2% halothane in oxygen and the head held in asteriotaxic clamp. The region around the lesion site was shaved and theskin over the skull slit in a sagtital plane with a scalpel and pulledback to leave the skull plates clear. A 0.5 mm diameter hole was drilledthrough the skull plate 3 mm to the right of bregma using an Arlecengraver and a lesion made into the cortex of the brain using a 19G 1½gauge syringe needle attached to a micrometer stage. The stage allowedaccurate directional control and a precise 2 mm penetration depth whichkept the lesion within the cortex and well above the corpus callosum.

With the animal prepared, 1 Oml of ice cold Pluronic F-127 gel (BASF)containing connexin 43 specific ODN (or a control ODN) was sucked into aprecooled 19G 1½ gauge syringe needle filed off so as to have a flattip. The syringe needle was attached to a volumetric pipette via a cutdown yellow pipette tip. The gel then set in the needle as it warmed toroom temperature. The needle with the gel plug at its tip wastransferred to a 1 ml syringe containing PBS and a sleeve slipped overthe needle shaft so that the needle tip could be lowered into the lesionwith the sleeve (coming up against the skull) preventingoverpenetration. Gentle pressure on the syringe plunger “popped” the gelplug out of the needle into the lesion. The wound was then treated withhydrogen peroxide to stop bleeding and the skin sutured back into place.Animals were carefully monitored and left until ready for sacrifice 24hours, 48 hours or 12 days later.

Frozen Sectioning

Animals were sacrificed using Nembutal (pentobarbitone sodium, Virbac)and decapitated. The brains were removed intact and immediately frozenin dry ice snow and stored at −80° C. until ready for sectioning. Serialcryosections (30 mm sections) were taken from front to rear (coronalplane), the sections dry mounted onto chrome alum treated slides, andstored for histochemistry or immunohistochemistry at −80° C. The firstand last section of each lesion was recorded so that the mid-pointsections of the lesion were clearly identified.

Histochemistry

For haemotoxylin and eosin staining sections were hydrated through adescending series of alcohols (absolute, 2×95%, 1×70% and water) andstained in Gill's haemotoxylin for 4 minutes. The sections were thenwashed in water, dipped in Scott's water and rewashed in water. Theywere then stained for 30 seconds in Moore's buffered eosin. The sectionswere washed once more in water before dehydration through a series ofalcohols (2×95%, 1× absolute), 50:50 alcohol:xylol and dipped in xylene.The sections were then mounted using Histomountä mounting medium.

For Nissl staining, sections were dehydrated in an ascending gradedseries of alcohols (75%, 95%, 3×100%), five minutes in each, anddefatted in xylene for five minutes. The sections were then rehydratedby descending through the same series of alcohols and washed in water.The sections were then placed in a Nissl staining solution (5 ml of a 2%aqueous Cresyl violet stock solution, 90 ml of a 6% glacial acetic acidin water solution, 10 ml of a 1.35% sodium acetate solution) for 10minutes. The sections were then quickly dehydrated in a series ofascending alcohols for 5 minutes at 75%, then 2 minutes each at 95% and3×100%, three charges of xylene for 10 minutes each. They were thencoverslipped with Histomountä mounting medium.

Immunohistochemistry

Frozen sections were first allowed to come back up to room temperaturein PBS. They were then permeabilised in methanol for two minutes, rinsedin PBS and transferred to a solution of 0.1 M lysine and 0.1% Triton-X100 in PBS for blocking over 30 min. Two washes in PBS, each of twominutes, followed. PBS was removed and 50 ml per section of primaryantibody was applied.

Immunohistochemistry was carried out with primary antibodies againstconnexin 43, Neuronal-Nuclei (vertebrate specific nuclear protein NeuN)and GFAP (glial fabrillary acidic protein). The following antibodieswere used:

-   Rabbit anti-Cx 43 (Gourdie et al., (1991)) at a concentration of    1:300.-   Mouse anti-Cx 43 (Chemicon International, Inc.) at a concentration    of 1:100.-   Rabbit anti-rat GFAP (DAKO, Z0334), at a concentration of 1:1000.-   Mouse anti-Neuronal Nuclei (Chemicon International, Inc.) at a    concentration 1:1000.

For connexin and GFAP labelling sections were incubated overnight at 4°C. They were then washed three times 15 minutes in PBS on an orbitalshaker. Following this, excess PBS was removed and 50 ml per section ofAlexaä 488 anti-rabbit IgG (Molecular Probes, Oregon, USA) was appliedat a concentration of 1:200. For monoclonals and double labelling a CY3(Chemicon, 132C) anti-mouse secondary antibody was used. Sections wereincubated in the dark for two hours at room temperature followed bythree washes of 15 minutes in PBS. For mounting excess PBS was removedfrom the slides and one or two drops of Citifluor (glycerol/PBSsolution) anti-fade medium was applied. A coverslip was lowered onto thesections and sealed with nail varnish. For Neuronal-N labelling thesecondary antibody was a biotinylated Goat anti-mouse followed by anavidin linked HRP and DAB reaction (Sigma ExtrAvidin or DAKO Quickstainkit).

Imaging and Analysis

Immunofluorescent labelling was carried out using a Leica TCS 4Dconfocal laser scanning microscope. Double labelled images weresubsequently combined using the Leica Combine function or in AdobePhotoshop. Haemotoxylin and eosin, and Nissl stained samples orNeuronal-N labelled sections were captured using a Kontron (Zeiss)Progress 3008 digital camera and lesion areas analysed using MetaMorph(Universal Imaging Corp). Lesion areas were analysed for the middlesection of each lesion.

Results

The well documented spread of brain lesions in the first 24-48 hoursafter trauma occurred in our control gel experiments and all lesions,controls and antisense treated, tended to spread near the outer edgewhere the gel is less likely to sit after loading. However, controllesions spread downwards into the corpus callosum and sideways to formragged, spreading edges (FIGS. 1A-1D and 2A-2B). Examination ofNeuronal-N antibody labelled tissues reveals neuronal death occurringwell back from the lesion edge, with areas of Nissl staining in which noviable neurons remain. This spread occurs predominantly within 24 hours(FIGS. 1A-1D and 2A-2B), continuing up to 48 hours after lesioning. Thisis especially apparent in FIG. 2 where neuronal death is evident within24 hours well back from the lesion edge into otherwise normal lookingtissue, and the lesion has spread right down into the corpus callosum.In contrast, the better connexin 43 antisense treated lesions remainconfined to the original lesion site and have clearly defined baselevels (FIGS. 3A-3B and 4A-4B). Neuronal N labelling colocalises withNissl stained tissue and none of the connexin 43 antisense treatedlesions spread through the corpus callosum. Neuronal-N labelling showsneuronal survival right up to the edge of the original needle tractlesion. Surviving neurons around these lesions often define sharpboundaries marking the edge of the needle tract (FIGS. 3A-3B and 5).More tissue remains viable within the lesion itself after antisensetreatment; in control lesions cell death leads to tissue loss within thelesion area (compare control lesion in FIG. 2A-2B at 24 hours withantisense treated lesions in FIGS. 3A and 3B and 4A and 4B at 48 hours).

While antibody labelling of glial fibrillary acidic protein (GFAP) showssome increased astrocyte activation at the edges of lesions, connexin 43protein levels are clearly reduced at many places along the edge ofantisense treated lesions, particularly the basal and medial edges (FIG.6) compared with controls (FIG. 7). In some areas the only connexin 43labelling remaining 24 hours after connexin 43 specific antisensetreatment is in blood vessel walls despite raised GFAP levels (FIG. 6).In general, connexin 43 labelling around antisense treated lesioncollocalises to a much lesser extent with GFAP labelling than incontrols in which over half of the connexin 43 labelling is astrocyterelated. Other connexin levels (connexins 26 and 32) did not appear tobe altered by the connexin 43 specific antisense treatments.

36 animals were lesioned. Cross sectional area (central slice of thelesion volume in a coronal plane) was analysed for 21 animals. Theresults are shown in Table 2.

TABLE 2 Cross sectional areas of lesions treated with control andconnexin 43 specific oligodeoxynucleotides, left empty, or treated withgel only. Measurements are for animals measured after 24 hours, 48 hoursand 12 days. Two sets of figures are included-measurements of the entirelesion, and measurements from 1 mm below the surface. In analysis of thesecond group the largest DB 1 treated lesion (brackets) is excluded asit falls outside 3 standard deviations from the mean for this group.Note that the rat brain does heal (unlike other species) and 12 dayslesion measurements do not represent the original extend of lesionspread. DB 1 is anti connexin 43 (SEQ. ID. NO: 2) treated HB3 is randomoligo (SEQ. ID. NO: 10) and appears to be toxic 24 hours 48 hours 12days Entire Lesion: (measurements in square mm) DB1 2.42; 3.16; 3.78;5.57 3.7; 6.05; 2.91; 2.79; 2.86 3.41; 4.53 HB3 7.14 13.19 Gel/empty5.04; 4.48 3.96; 3.41; 3.56; 5.91 2.58; 3.3 Lesions from 1 mm down:(this is considered a more accurate measure as all lesions tend tospread at the outer lip indicating that the treatment gel has settled inthe bottom of the lesion and/or the outer cortex has been damaged whendrilling the skull or inserting the gel loading needle. DB1 0.91; 1.13;2.12; 2.41 (3.38); 0.99; 1.54; 0.47; 1.2 1.44; 1.08 HB3 5.9 5.6Gel/empty 3.2; 2.19 1.86; 1.5; 1.68; 2.17 1.07; 1.43

In the final analysis the lesion area from a line 1 mm below the outercortex edge was measured so as to exclude lesion spread at the outeredge where antisense treatments have little or no effect (owing to gelbeing injected into and settling at the bottom of lesions). Oneantisense treated animal falls more than three standard deviationsoutside the mean for this group and has been excluded. Mean lesion sizefor antisense treated lesions at 24 and 48 hours was 1.45 mm² (+/−0.55),for controls 2.1 mm² (+/−0.6). The four smallest (of 8 antisense treatedand 8 control lesions at 24 and 48 hours) were all connexin 43 antisensetreated, with the smallest control lesion 50% larger than these four.This data is also shown in graphical form in FIG. 8. By 12 daysregeneration occurs in the rat (but not in human brain tissue) and thelimits of lesion spread are not clearly defined.

Discussion

The Pluronic gel plug-antisense ODN method has been used to study theeffect of connexin 43 knockdown during astrocytosis which occursfollowing lesioning of the cerebral cortex of the mammalian brain. Inthe brain, release of toxins from dying neurons causes what is known asthe bystander effect, with the toxins spreading to neighbouring cellsthrough gap junction channels (Lin et al, (1998)). Underneurodegenerative conditions, slow release of toxins apparently leads toan upregulation of connexin 43 channels in astrocytes to enable thetransport and removal of the toxins to the blood stream. In cases ofsevere trauma however, this upregulation aids the spread of high toxinlevels to neighbouring neurons, killing them. Blocking of the connexin43 upregulation and knockdown of connexin 43 channels prevents thisspread leading to lesions up to 50% smaller in cross sectional area.This has significant implications in the management of ischeamic stroke,treatment of neurodegenerative diseases, and modulation of side effectsfrom surgical intervention.

Experiment 3

Introduction

The bystander effect in neural tissues whereby damaged neurons releasetoxins which spread and kill neighbouring cells is well documented.Experiment 2 shows that this effect can be reduced in the brain using anantisense oligodeoxynucleotide sustained release approach to knockdownthe gap junction protein connexin 43.

Another tissue of similar composition to the brain is the spinal cord inwhich the neural population is supported by populations of glial cells,including astrocytes which are responsible for the neuroprotectiveeffect by removing glutamate and excess calcium from the neuralenvironment. This experiment investigates the ability of theformulations of the invention to reduce the spread of spinal cordlesions.

Materials

Oligodeoxynucleotides were prepared with the following sequences:

(SEQ ID NO: 2) GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC  (connexin 43) (SEQ ID NO: 7) GAC AGA AAC AAT TCC TCC TGC CGC AAT TAC  (sense control) 

Methods

Wistar rats were anaesthetised and their spinal cord exposed. A standardhemisection lesion was then made in the cord and 5 ml of chilledPluronic gel, containing either antisense or sense ODN's to connexin 43(5 μM) was placed in the lesion. Applications were made blind. Theexposed cord was then recovered and the rat returned to its cage. Someanimals were sacrificed at 24 hours whereas others were maintained for12 days and two months in order to determine the extent of neuronalregeneration and the final size of the lesion. For axonal regenerationstudies the rats were anaesthetised and their axons severed prior totheir entry site to the spinal cord. A pellet of Horse radish peroxidase(HRP) was placed in the cut in order to retrogradely label the axonsover a 24 hour period. Next day the rats were sacrificed and theirspinal cords removed and fixed in 2% paraformaldehyde. Cords were thenpi-ocessed for cryosectioning and serial longitudinal 8 mm sections weretaken through the cords. Sections were then immunostained for eitherconnexins or GFAP along with propidium iodide as a nuclear marker, orprocessed to reveal the HRP.

Results

At 24 hours post lesion there was a marked difference between the spinalcord lesions treated with connexin 43 sense and antisense ODN's. Thesense lesions appeared no different from untreated controls whereas theantisense treated lesions appeared smaller and less inflamed (FIG. 9).

At 12 days HRP labelled axons could be seen in both sense and antisensetreated cords but in neither case did significant numbers ofregenerating axons cross the lesion. However, there was a markeddifference in lesion size with the antisense lesion appearingsignificantly smaller than the sense or untreated lesions.

Two months after lesioning the spinal cords HRP labelling ofregenerating axons revealed that they had failed to cross the lesionsite in both sense and antisense treatments. Lesion size wassignificantly smaller in antisense treated cords indicating asignificant reduction in secondary neuronal cell death.

Discussion

Using the formulations of the invention, the antisenseoligodeoxynucleotide knockdown of connexin 43 significantly reduces thelesion spread which occurs in the first 24-48 hours after spinal cordinjury. The knockdown of connexin 43 also reduces inflammation, furtheraiding in the neuroprotective effect, but there was no change in theability for neurons to grow back across the lesion site. Thus, antisensetreatment with connexin 43 specific oligodeoxynucleotides cannot aidregrowth of damaged neurons, but has a significant neuroprotectiveeffect reducing the spread of the insult.

Experiment 4

Introduction

To repair skin wounds a number of cell types, such as fibroblasts,endothelial cells and keratinocytes are activated to proliferate,migrate and lay down extracellular matrix to fill the wound.

Communication and intercellular signalling is a key feature of the woundhealing process. Extracellular signalling mechanisms are thought to bethe key players though it is also probable that intercellular signallingthrough the extensive networks of gap junction channels in the skinlayers may also have a role. Calcium waves spreading away from injuredcells through the epidermis may signal their damage. In normal woundhealing connexin levels start to fall within 6 hours and take up to 6days to recover. The roles that these changes play are not understoodbut one theory is that cells are released from their neighbours todivide rapidly, and then junctions reform to coordinate migration intoand over the wound site.

This experiment investigates the ability of the formulations of theinvention to effect wound healing.

Materials

Oligodeoxynucleotides were prepared with the following sequences:

(SEQ ID NO: 2) GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC   (connexin 43)(SEQ ID NO: 7) GAC AGA AAC AAT TCC TCC TGC CGC AAT TAC   (sense control)

Methods

Neonatal mice, CD1 strain, were anaesthetised with local anaesthetic byspray. A clean incision wound, 2 mm long, was then made along the lengthof both fore paws with an iridectomy knife. By making the wounds under adissecting microscope they can be made very reproducible in size. Theygenerally heal in 3-6 days. Carbon powder was dusted into the wounds inorder to mark them for subsequent identification of the wound site atlate time points—this does not affect the healing in any way. 5 ml ofchilled Pluronic gel, containing either Sense or Antisense ODN's wasthen applied to the wounds. The Pluronic gel is liquid between 0-4° C.but sets at higher temperature. Once applied to the wound the gel setsin place and acts as a slow release reservoir for the ODN's as well as amild surfactant, aiding the penetration of ODN's into the tissue.Application of Sense ODN's was made to one paw and Antisense to theother, alternating left and right between litters. Pups were warmedunder a lamp and then returned to their mother. Wounds were examineddaily and scored for quality of healing. Representative pups wereselected at 1 day, 5 day and 8 day post operation and their forelimbsphotographed before the pups were anaesthetised and perfused with 2%paraformaldehyde. The forelimbs were removed and immersion-fixed in 2%paraformaldehyde overnight and then processed for resin (1 day) or wax(2 days onward) histology.

Inflammation of the wound was assessed 24 hours after wounding. Resinsections through the wound are stained with Toluidine blue to revealnissl positive cells, neutrophils, which are the first cells to respondto injury. These can also be revealed using neutrophil specific markers.

Cell death and clearance is assessed by Tunel labelling to determine therate of clearance of apoptotic cells. Macrophage staining was used toshow the period of clearing up following cell death. These are carriedout days 3-5 post wounding.

Angiogenesis

Granulation is a feature of healing connective tissue and is cased bythe invasion of numerous capillaries. Macrophages are known to expresspotent angiogenic factors such as VEGF. The degree of vascularisation ismonitored with antibodies to VEGF receptors, anti-PCAM and anti-flt-1which are both good blood vessel markers. Contraction of this tissue isbrought about by the differentiation of wound fibroblasts into acontractile myofibroblast. After they have pulled the wound togetherthey die apoptotically and are removed by macrophages. These cells canbe revealed by smooth muscle actin specific antibodies and theirformation and removal followed.

Hyperinnervation

Sensory nerves are very sensitive to the signals released on woundingand show transient sprouting at the sites of adult wounds. However, inneonatal wounds this sprouting is more profuse and results in permanenthyperinnervation. Whilst it is not clear what these signals are it islikely that they are released from inflammatory macrophages.Hyperinnervation is maximal at 7 d post wounding and nerve distributioncan be revealed using PGP 9.5 antibody against neurofilaments.

Scarring is normally assessed weeks or months after closure of thewound. However, a reasonable assessment can be made 12 days afterwounding. Sections through the wounds are stained with the collagenstain Picrosirus Red and examined on a confocal microscope to determinethe collagen density and orientation at the wound site.

Results

1 Day

At 24 hours after wounding marked differences were apparent between thesense and antisense treated limbs. Sense treated wounds looked nodifferent from untreated with a normal spectrum of healing grades andrates (FIG. 10). Antisense treated limbs were markedly different fromthe controls, they appeared to be less inflamed and the healing rate wasgenerally faster.

Resin sections of representative limbs stained with a nissl stainrevealed significantly less neutrophils cells indicating a less inflamedtissue (FIG. 11).

5 Days

By days after wounding scabs had started to fall off. At this stage mostof the antisense treated wounds appeared to be smaller than the sensetreated with either small scabs or less prominent scarring (FIG. 12).

8 Days

8 days after wounding the limbs had grown hair. Sense treated woundswere still visible being demarcated by a lack of hair around the woundsite. Antisense treated wounds were mostly invisible being covered bynormal hair growth. This difference in hair growth indicates reducedscarring has occurred in the antisense treated wounds (FIG. 13).

Conclusions

Application of connexin 43 antisense ODN's to a wound has a markedaffect on the healing process. The first noticeable effect is areduction in the inflammation of the wounds which is noticeable insections which show a much lower inflammatory response in terms oflevels of neutrophils. As healing progresses, antisense treated woundsheal faster and with less scarring than control lesions.

This reduction in inflammatory response and subsequent improved healingis possibly owing to reduced neutrophil communication and to a speedingup of natural healing processes. The antisense ODN's can reduce connexinexpression in 4-8 hours so they will not have an effect on the initialsignalling of wounding but play a role in the secondary signallingevents. It is interesting to note that neutrophils which invade inresponse to the wounding normally express large amounts of connexin 43.It is also possible that they form gap junctions with other cells in thewound and communicate with them. Reduction in this form of communicationmay result in a reduction of secreted factors from the neutrophils andmay reduce cell death in the wound as well as granulation andhyperinnervation. It is also known that under normal conditions connexinprotein levels (connexins 26. 31.1 and 43) are reduced in both theepithelial and subdermal layers of wounds starting within 6 hours, andremaining lowered for up to 6 days. The antisense approach may speed upthis initial protein reduction by blocking translational processes asprotein removal from the membrane is occurring. Certainly, the effectsof connexin 43 knockdown immediately following wounding has markedeffects on reducing inflammatory levels and increasing healing rates.

Experiment 5

Introduction

The inflammation and secondary cell death that follows burning is ofmajor concern. Victims of severe burns over a high percentage of theirbody often die one or two days after trauma. This experimentinvestigates the ability of the formulations of the invention tobeneficially affect the burn recovery process.

Materials

(SEQ ID NO: 2) GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC.  (connexin 43) (SEQ ID NO: 7) GAC AGA AAC AAT TCC TCC TGC CGC AAT TAC   (sense control)

Oligodeoxynucleotides were prepared with the following sequences:

Methods

Reproducible burns are delivered to moistened skin, and Pluronic gelcontaining antisense ODN's injected subdermal to the burn. A series ofburns were made using a soldering iron to the left and right sides ofthe skull of six newborn mice. The burns on one side of the head weretreated with connexin 43-specific ODN in Pluronic gel and those on theother side with sense control ODN in Pluronic gel.

Results

After 24 hours, all six connexin 43 ODN treated burns showed lowerlevels of inflammation compared with the control burns. Thesedifferences were marked (data not shown).

Utility

Thus, in accordance with the invention, there are provided formulationsby which cell-cell communication can be downregulated in a transient andsite-specific manner. The formulations therefore have application inmethods of therapy and in cosmetic treatments.

The delivery of the ODN component of the formulation for an extendedperiod (24 hours or longer) is a particular advantage in treatingneuronal damage. This is because, in most instances of direct physicalneuronal insult, neuronal cell loss extends well beyond the site ofactual injury to the surrounding cells. This secondary neuronal cellloss occurs within 24 hours of the original injury and is mediated byjunction gap cell-cell communication. Downregulation of connexin proteinexpression therefore blocks or at least downregulates communicationbetween the cells and minimises secondary neuronal cell damage.

Equally, in instances of other tissue damage (particularly wounds) theformulations of the invention have been found effective in bothpromoting the wound healing process, reducing inflammation and inminimising scar formation. The formulations therefore have clear benefitin the treatment of wounds, whether the result of external trauma(including burns) or surgical intervention.

It will further be appreciated that the above description is provided byway of example only and that modifications can be made, both in terms ofthe specific ODN's and pharmaceutically acceptable carriers or vehiclesemployed without departing from the scope of the present invention.

TABLE 3 (SEQ ID NO: 12) 1atgggtgact ggagcgcctt aggcaaactc cttgacaagg ttcaagccta ctcaactgct 61ggagggaagg tgtggctgtc agtacttttc attttccgaa tcctgctgct ggggacagcg 121gttgagtcag cctggggaga tgagcagtct gcctttcgtt gtaacactca gcaacctggt 181tgtgaaaatg tctgctatga caagtctttc ccaatctctc atgtgcgctt ctgggtcctg 241cagatcatat ttgtgtctgt acccacactc ttgtacctgg ctcatgtgtt ctatgtgatg 301cgaaaggaag agaaactgaa caagaaagag gaagaactca aggttgccca aactgatggt 361gtcaatgtgg acatgcactt gaagcagatt gagataaaga agttcaagta cggtattgaa 421gagcatggta aggtgaaaat gcgagggggg ttgctgcgaa cctacatcat cagtatcctc 481ttcaagtcta tctttgaggt ggccttcttg ctgatccagt ggtacatcta tggattcagc 541ttgagtgctg tttacacttg caaaagagat ccctgcccac atcaggtgga ctgtttcctc 601tctcgcccca cgagaaaac catcttcatc atcttcatgc tggtggtgtc cttggtgtcc 661ctggccttga atatcattga actcttctat gttttcttca agggcgttaa ggatcgggtt 721aagggaaaga gcgaccctta ccatgcgacc agtggtgcgc tgagccctgc caaagactgt 781gggtctcaaa aatatgctta tttcaatggc tgctcctcac caaccgctcc cctctcgcct 841atgtctcctc ctgggtacaa gctggttact ggcgacagaa acaattcttc ttgccgcaat 901tacaacaagc aagcaagtga gcaaaactgg gctaattaca gtgcagaaca aaatcgaatg 961gggcaggcgg gaagcaccat ctctaactcc catgcacagc cttttgattt ccccgatgat 1021aaccagaatt ctaaaaaact agctgctgga catgaattac agccactagc cattgtggac 1081cagcgacctt caagcagagc cagcagtcgt gccagcagca gacctcggcc tgatgacctg 1141gagatctag

REFERENCES

Becker, D. L., Evans, W. H., Green, C. R., Warner, A. (1995): Functionalanalysis of amino acid sequences in connexin 43 involved inintercellular communication through gap junctions. J. Cell Sci. 108,1455-1467.

Becker, D. L., McGonnell, I., Makarenkova, H. P., Patel, K., Tickle, C.,Lorimer, J. and Green, C. R. (1999). Roles for a1 connexin inmorphogenesis of chick embryos revealed using a novel antisenseapproach. Devel. Genetics, 24, 33-42.

Cotrina, M. L., Kang, J., Lin, J. H-C., Bueno, E., Hansen, T. W., He,L., Lie, Y. and Nedergaard, M. (1998). Astrocytic gap junctions remainopen during ischemic conditions. J. Neurosci., 18, 2520-2537.

Giaume, C. and McCarthy, K. D. (1996). Control of gap junctionalcommunication in astrocytic networks. TINS, 19, 319-325.

Gourdie, R. G., Green, C. R., Severs, N. J. (1991). Gap junctiondistribution in adult mammalian myocardium revealed by an anti-peptideantibody and laser scanning confocal microscopy. J. Cell Sci. 99: 41-55.

Green, C. R., Bowles, L., Crawle, A., Tickle C. (1994): Expression ofthe connexin 43 gap junctional protein in tissues at the tip of thechick limb bud is related to epithelial-mesenchymalinteractions thatmediate morphogenesis. Devel. Biol., 161, 12-21.

Lin, J. H., Weigel, H., Cotrina, M. L., Liu, S., Bueno, E., Hansen, A.J., Hansen, T. W., Goldman, S. and Nedergaard, M. (1998).Gap-junction-mediated propogation and amplification of cell injury.Nature Neurosci., 1, 431-432.

Neckers, L., Whitesell, L. (1993): Anti-sense technology: biologicalutility and practical considerations. Am. J. Physiol. 265 (lung cell molphysiol), LI-LI2.

Simons, M., Edelman, E. R., DeKeyser, J. L., Langer, R., Rosenberg, R.D. (1992): Anti-sense c-myb oligonucleotides inhibit intimal arterialsmooth muscle cell accumulation in vivo. Nature, 359, 67-70.

Stein, C. A. (1992): Anti-sense oligodeoxynucleotides-promises andpitfalls, Leukemia 6, 967-974. Wagner, R. W. (1994): Gene inhibitionusing anti-sense oligodeoxynucleotides, Nature, 372, 333-335.

What is claimed is:
 1. A pharmaceutical composition comprising a connexin 26 antisense polynucleotide and a nonionic polyoxyethylene-polyoxypropylene copolymer, wherein said connexin 26 antisense polynucleotide is an oligonucleotide provided in an amount sufficient for treatment of a wound.
 2. A pharmaceutical composition according to claim 1, which is formulated for topical administration.
 3. A pharmaceutical composition according to claim 1, which is formulated for sustained release administration.
 4. A pharmaceutical composition according to claim 1, formulated with a connexin antisense polynucleotide in an amount sufficient to downregulate the expression of a connexin protein in a mammal.
 5. A pharmaceutical composition according to claim 1, formulated with a connexin antisense polynucleotide in an amount sufficient to decrease cell death in a tissue of a mammal.
 6. A pharmaceutical composition according to claim 1, formulated with a connexin antisense polynucleotide in an amount sufficient to decrease inflammation in a tissue of a mammal.
 7. A pharmaceutical composition according to claim 1, formulated with a connexin antisense polynucleotide in an amount sufficient for treatment of a wound in a tissue of a mammal.
 8. A pharmaceutical composition of claim 1, wherein said connexin antisense polynucleotide is about 12 to about 40 nucleotides in length.
 9. A pharmaceutical composition of claim 1, wherein said connexin antisense polynucleotide is about 20 nucleotides in length.
 10. A pharmaceutical composition of claim 1, wherein said connexin antisense polynucleotide is about 30 nucleotides in length.
 11. A pharmaceutical composition of claim 1, wherein said connexin antisense polynucleotide is about 40 nucleotides in length.
 12. A pharmaceutical composition of claim 8, wherein said connexin antisense polynucleotide comprises SEQ ID NO:
 4. 13. A pharmaceutical composition of claim 1, formulated as a cream, ointment, gel, emulsion, or paint.
 14. A pharmaceutical composition of claim 13, formulated as a gel.
 15. A pharmaceutical composition of claim 14, formulated as a gel for topical administration.
 16. A pharmaceutical composition of claim 1, wherein the polynucleotide is formulated for parenteral, intramuscular, intracerebral, intravenous, subcutaneous or transdermal administration.
 17. A pharmaceutical composition comprising a connexin 26 antisense polynucleotide in an amount effective for treatment of a wound in a mammal and a nonionic polyoxyethylene-polyoxypropylene copolymer, wherein said connexin 26 antisense polynucleotide is an oligonucleotide.
 18. A pharmaceutical composition according to claim 1 or 17, wherein said nonionic polyoxyethylene-polyoxypropylene copolymer is in the form of a gel.
 19. A pharmaceutical composition according to claim 1 or 17, wherein said nonionic polyoxyethylene-polyoxypropylene copolymer is Pluronic F-127.
 20. A pharmaceutical composition according to claim 17, which is formulated for sustained release of the antisense polynucleotide.
 21. A pharmaceutical composition according to claim 1 or 17, which further includes a surfactant or transdermal penetration agent.
 22. A pharmaceutical composition according to claim 1 or 17, wherein said composition is formulated with an isotonic saline solution.
 23. A pharmaceutical composition according to claim 1 or 17, wherein said composition is formulated with phosphate buffered saline.
 24. A pharmaceutical composition according to claim 18, wherein the composition is formulated for topical administration.
 25. A pharmaceutical composition according to claim 19, wherein the composition is formulated for topical administration.
 26. A pharmaceutical composition according to claim 20, wherein the composition is formulated for topical administration.
 27. A pharmaceutical composition according to claim 21, wherein the composition is formulated for topical administration.
 28. A pharmaceutical composition according to claim 1 or 17, wherein said nonionic polyoxyethylene-polyoxypropylene copolymer is poloxamer
 407. 29. A pharmaceutical composition according to any of claims 1, 2, 4-28, wherein said polynucleotide is an unmodified phosphodiester oligomer.
 30. A pharmaceutical composition according to any of claims 1, 2, 4-28, wherein said antisense polynucleotide is chemically modified. 